The lab continues to use in vitro systems and model organisms to study the function of PKD1, PKD2 and PKHD1, the genes responsible for the most common forms of autosomal dominant and autosomal recessive polycystic kidney disease. To further investigate a metabolic phenotype in Pkd1 mutant cells that we had reported last year, we have analyzed metabolic flux by mass spectroscopy of WT vs mutant cells treated with 13C-labeled glucose and confirmed that Pkd1 inactivation results in a mild, but detectable, shift in metabolite utilization. We also found that mutant cells loaded with labeled lipids had lipid droplets that were larger and more numerous than in controls, suggesting that lipids were not utilized as efficiently as in controls. Our studies of Pkd1 mutant cell lines and freshly isolated cells from Pkd1 mutant kidneys reveal differences in both mitochondrial function and morphology. We are currently pursuing studies that seek to link PC1 function directly to these properties using a variety of biochemical and cell biological methodologies. We also have initiated a collaboration with the Xu laboratory to study the function of the PC1 C-terminus in Drosophila. Our current working hypothesis is that PKD may be a disease where altered cellular metabolism is a primary component of the pathobiology. We have expressed dual-tagged forms of PC1 in fibroblast and epithelial cell lines and identified novel patterns of expression. To better examine these properties in vivo, we have collaborated with the NHLBI mouse transgenic core to make a mouse line with an EGFP knocked into the C-terminus of PC1 by CRISPR. The first mouse verified to have correct incorporation of the construct failed to transmit the allele so we have generated a new mouse that has just begun breeding. We have collaborated with the NIDDK and NHLBI Mass Spectrometry cores to identify putative PC1-binding partners. One of the interactors of interest was previously identified as being differentially expressed in cystic tissue. Studies are underway to better understand these observations and the link to PC1. Last year we had described the results of studies that revealed a novel link between the PKHD1 gene product, FPC (fibrocystin/polyductin), and multiple members of the C2 WWW HECT domain E3 family of ubiquitin ligases. These data provide a mechanistic explanation for both the cellular effects and important in vivo phenotypic abnormalities observed in mice and humans that result from Pkhd1/PKHD1 mutation. The manuscript describing these findings has now been published. The C-terminus of FPC is reported to include a number of essential functional domains including a ciliary targeting sequence, a nuclear localization signal and a polycystin-2 (PC2) binding site and to traffic to the nucleus after cleavage. We collaborated with the Watnick lab (U. of Maryland) to generate a novel Pkhd1 mouse model with an HA-epitope knocked-in to the C-terminus to allow tracking in vivo and lox P sites flanking the last exon that encodes most of the C-terminus (Pkhd1HA-Flox). In characterizing tissues from the model, we have for the first time identified the previously predicted cleavage products in tissue. We also find that PC2 fails to co-precipitate with FPC from kidney samples. Immunofluorescence studies show that FPC is primarily present in a sub-apical location in kidney, biliary duct and pancreatic ducts, partially overlapping with the Golgi. Contrary to previous studies, we cannot detect the protein in the primary cilia. After Cre-mediated deletion, we remarkably find that homozygous Pkhd1 67 mice are completely normal, in contrast to Pkhd1del3-4 homozygotes in the same strain that have both pancreatic and biliary disease. These studies show that Pkhd1HA-Flox is a valid model to track Pkhd1-dervied products containing the C-terminal and that exon 67 is not essential for FPC function in mice. A manuscript describing these findings has just been published. We have also continued to characterize a mouse line generated in collaboration with the NHLBI mouse core that has a CRISPR-induced deletion within a gene found to be essential for PC2 trafficking in Drosophila. We found normal birth rates and no obvious abnormalities thus far in a cohort of mice allowed to age. There is a highly homologous second gene that may be providing compensation.